Molecular Mechanisms Regulating BAFF and APRIL Receptor Expression in B Cells: Promoter Structure and Epigenetics

Abstract Throughout differentiation, mature B cells express distinct combinations of the BAFF and APRIL receptors, BAFF-R, TACI, and BCMA. The patterns of B lineage cell receptor expression reflect their stage of differentiation and impart the ability to respond to ligands, in some cases delivering a powerful anti-apoptotic signal. B cell malignancies arise from each stage of differentiation, typically exhibit the patterns of receptor expression that reflect their cell of origin, and have been shown to exploit the generally anti-apoptotic effects of BAFF and/or APRIL. For example, there is evidence for a role for BAFF in mature B cell cancers, including B cell chronic lymphocytic leukemia (B-CLL). As the majority of circulating CLL B cells are quiescent cells, prolonged survival is a significant hallmark, a trait that signals through BAFF-R could initiate or reinforce. Therapeutic strategies designed to interrupt this pro-survival pathway have thus far primarily focused on blocking ligand binding. Therapeutic modalities impacting receptor expression may be similarly effective. However, despite the apparent precise activation stage-dependent orchestration of B cell BAFF-R, TACI, and BCMA expression, the genetic mechanisms regulating expression of the three receptors remain undefined, and the question of whether each receptor governs expression of the other two remains unanswered. In agreement with previous studies in our own lab and others, analyses of the receptor profiles of CLL B cells continue to show BAFF-R surface expression, albeit at lower levels than seen on normal peripheral B cells, and the curious variable presence of BCMA and TACI. Similarly, multiple myeloma (MM) plasma cells (PCs), like normal PCs, uniformly lack BAFF-R expression, express BCMA, and variably express TACI. Our current study explores the mechanisms of receptor regulation in B cells, with an emphasis on BAFF-R, the receptor that is most consistently expressed on the CLL B cell population and that has the most clearly defined survival function. We began by analyzing the BAFF-R gene’s genomic context. We identified a possible regulatory element 2 kb upstream of the first exon with significant homology across seven mammalian species that overlapped a cluster of B cell lineage transcription factor binding sites, and, thus, we called the 2.5 kb directly upstream of the gene the putative BAFF-R promoter. We cloned the region and created promoter-reporter vectors in which the full-length promoter and 0.5 kb 5’ truncations thereof drive firefly luciferase production. While studies of primary B cells continue, studies with malignant B cell lines suggest that we have successfully cloned a powerful positive regulatory region. Specifically, B cell lines that express surface BAFF-R show positive inductions of firefly to control renilla luciferase activity in all of the promoter constructs over the empty construct with the greatest promoter activity in the longest constructs: 6-, 18-, and 3-fold inductions with the 2.5 kb promoter in RAJI, Loukes, and MEC-1 cells, respectively. To further test the promoter specificity, we transfected malignant PC lines, ALMC-1, ALMC-2, and KAS-6/1, which are negative by RT-PCR and surface staining for BAFF-R. These lines showed little promoter activity over baseline, with fold inductions between 0.5 and 2.5 for all of the promoter constructs. These results suggest that the MM lines no longer express the transcription factors required to drive BAFF-R expression and underscore our conclusion that we have identified the BAFF-R promoter. At the same time, investigations into epigenetic modification may reveal a crucial level of control. The transcriptional start site of the BAFF-R gene falls within a region of high CG content, and may be a possible CpG island. Upon treatment with the methyltransferase inhibitor, 5-azacytidine, primary blood B cells and MM cells showed no change in receptor expression. However, in CLL B cells, treatment of cultured cells caused a slight (9%) decrease in BAFF-R expression and prevented TACI upregulation in cells stimulated with CpG (a 76% increase fell to 36%). This evidence suggests that methylation indirectly suppresses expression of BAFF-R and TACI. It is essential to understand the regulation of survival receptors critical to normal B lineage cell survival, which may also be crucial for their malignant counterparts, in order to target those mechanisms as therapy.